Neonatal hypoxic-ischemic encephalopathy (HIE) from birth asphyxia causes persistent and severe neurologic disabilities, even in patients who receive therapeutic hypothermia. We found in clinical studies that white matter injury on MRI persists after hypothermic treatment. Thus, hypothermia is not fully protective. White matter injury is a prominent yet understudied component of the neurologic disabilities observed in neonates who receive hypothermia for HIE. Therapeutic adjuncts that protect the white matter might reduce the risk of permanent neurologic injury in HIE. Hypoxia-ischemia (HI) in neonatal pig, which has human-like white matter tracts, produces brain damage similar to that of full-term human newborns with HIE, including the white matter injuries observed in clinical studies. Our model includes clinically relevant whole-body hypothermia, rewarming at 0.5C/h, sedation, continuous hemodynamic monitoring, ventilator support, and correction of blood gas and electrolyte abnormalities to mimic clinical neonatal intensive care. Preliminary data suggest that insufficient proteasome function mediates persistent white matter injury after HI and hypothermia. We postulate that white matter proteasome insufficiency causes a failure to clear oxidatively damaged proteins, causing oligodendrocyte apoptosis, potential disruption of oligodendrocyte precursor maturation, myelin and axonal injury, and white matter volume loss after HI and hypothermia. We will elucidate the proteasome?s role in white matter injury after whole-body HI and overnight hypothermia in neonatal swine. White matter injury and oligodendrocyte biology will be studied with neuropathology (including oligodendrocyte precursor maturation, stereology, cell death, and electron microscopy) and biochemistry (including protein post-translational modification and proteasome composition and activity) through 1 month recovery after HI. T- maze neurocognitive behavior testing with neuropathology correlation will provide a functional outcome. We developed new methods to genetically modulate proteasome activity in distinct, targeted regions of white matter in neonatal pig forebrain using virus-mediated enforced expression of a proteasome activator subunit or proteasome inhibition with short hairpin small interfering RNA. We will also use a small molecule proteasome inhibitor to determine whether proteasome inhibition aggravates white matter injury. Moreover, we will test the potential of the drug oleuropein to protect white matter. Oleuropein is a readily bioavailable compound with proteasome activating properties and few clinical side effects. An intravenous oleuropein dosing regimen will be used that protects oligodendrocytes and myelin, increases proteasome expression, and promotes clearance of oxidized proteins after HI and hypothermia. We will identify whether oleuropein acts on the standard proteasome or the immunoproteasome. Cultured human oligodendrocyte experiments will validate the proteasome as a therapeutic target and oleuropein?s actions after oxygen glucose deprivation. This project will advance the neonatal HI and cell biology fields by investigating novel mechanisms by which proteasome insufficiency mediates hypothermia-resistant injury in white matter. We will discover whether proteasome activation is a relevant therapeutic adjunct to hypothermia to protect white matter and improve neurologic outcomes in HIE.